B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 54
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Note that for every 5.91kJ/mole decrease in standard free energy,the equilibrium constant increases by afactor of 10 at 37°C.The equilibrium constant here has units ofliters/mole; for simple binding interactionsit is also called the affinity constant orassociation constant, denoted Ka. Thereciprocal of Ka is called the dissociationconstant, Kd (in units of moles/liter).140Chapter 3: Proteinsof a few noncovalent bonds can have a striking effect on a binding interaction,as shown by the example in Figure 3–45. (Note that the equilibrium constant, asdefined here, is also known as the association or affinity constant, Ka.)We have used the case of an antibody binding to its ligand to illustrate theeffect of binding strength on the equilibrium state, but the same principles applyto any molecule and its ligand. Many proteins are enzymes, which, as we nowdiscuss, first bind to their ligands and then catalyze the breakage or formation ofcovalent bonds in these molecules.Consider 1000 molecules of A and1000 molecules of B in a eukaryoticcell.
The concentration of both willbe about 10–9 M.If the equilibrium constant (K )for A + B AB is 1010, then one cancalculate that at equilibrium therewill be270Enzymes Are Powerful and Highly Specific CatalystsMany proteins can perform their function simply by binding to another molecule.An actin molecule, for example, need only associate with other actin moleculesto form a filament. There are other proteins, however, for which ligand binding isonly a necessary first step in their function. This is the case for the large and veryimportant class of proteins called enzymes. As described in Chapter 2, enzymesare remarkable molecules that cause the chemical transformations that make andbreak covalent bonds in cells.
They bind to one or more ligands, called substrates,and convert them into one or more chemically modified products, doing this overand over again with amazing rapidity. Enzymes speed up reactions, often by afactor of a million or more, without themselves being changed—that is, they actas catalysts that permit cells to make or break covalent bonds in a controlled way.It is the catalysis of organized sets of chemical reactions by enzymes that createsand maintains the cell, making life possible.We can group enzymes into functional classes that perform similar chemicalreactions (Table 3–1). Each type of enzyme within such a class is highly specific,270ABmolecules molecules730ABmoleculesIf the equilibrium constant is a littleweaker at 108, which representsa loss of 11.9 kilojoule/mole ofbinding energy from the exampleabove, or 2–3 fewer hydrogenbonds, then there will be915915ABmolecules molecules85ABmoleculesFigure 3–45 Small changes in thenumber of weak bonds can have drasticeffects on a binding interaction.
Thisexample illustrates the dramatic effect ofthe presence or absence of a few weaknoncovalent bonds in a biological context.MBoC6 m3.44/3.41TABLE 3–1 Some Common Types of EnzymesEnzymeReaction catalyzedHydrolasesGeneral term for enzymes that catalyze a hydrolytic cleavage reaction; nucleases and proteases aremore specific names for subclasses of these enzymesNucleasesBreak down nucleic acids by hydrolyzing bonds between nucleotides. Endo– and exonucleasescleave nucleic acids within and from the ends of the polynucleotide chains, respectivelyProteasesBreak down proteins by hydrolyzing bonds between amino acidsSynthasesSynthesize molecules in anabolic reactions by condensing two smaller molecules togetherLigasesJoin together (ligate) two molecules in an energy-dependent process.
DNA ligase, for example, joinstwo DNA molecules together end-to-end through phosphodiester bondsIsomerasesCatalyze the rearrangement of bonds within a single moleculePolymerasesCatalyze polymerization reactions such as the synthesis of DNA and RNAKinasesCatalyze the addition of phosphate groups to molecules. Protein kinases are an important group ofkinases that attach phosphate groups to proteinsPhosphatasesCatalyze the hydrolytic removal of a phosphate group from a moleculeOxido-ReductasesGeneral name for enzymes that catalyze reactions in which one molecule is oxidized while theother is reduced. Enzymes of this type are often more specifically named oxidases, reductases, ordehydrogenasesATPasesHydrolyze ATP. Many proteins with a wide range of roles have an energy-harnessing ATPase activityas part of their function; for example, motor proteins such as myosin and membrane transportproteins such as the sodium–potassium pumpGTPasesHydrolyze GTP.
A large family of GTP-binding proteins are GTPases with central roles in theregulation of cell processesEnzyme names typically end in “-ase,” with the exception of some enzymes, such as pepsin, trypsin, thrombin, and lysozyme, that werediscovered and named before the convention became generally accepted at the end of the nineteenth century. The common name of an enzymeusually indicates the substrate or product and the nature of the reaction catalyzed. For example, citrate synthase catalyzes the synthesis of citrateby a reaction between acetyl CoA and oxaloacetate.PROTEIN FUNCTION141rate of reactionVmax0.5VmaxKmsubstrate concentrationcatalyzing only a single type of reaction.
Thus, hexokinase adds a phosphate groupto D-glucose but ignores its optical isomer L-glucose; the blood-clotting enzymethrombin cuts one type of blood protein between a particular arginine and itsadjacent glycine and nowhere else, and so on. As discussed in detail in Chapter 2,enzymes work in teams, with the product of one enzyme becoming the substrateMBoC6 m3.45/3.42for the next. The result is an elaborate network of metabolicpathways that provides the cell with energy and generates the many large and small molecules thatthe cell needs (see Figure 2–63).Substrate Binding Is the First Step in Enzyme CatalysisFor a protein that catalyzes a chemical reaction (an enzyme), the binding of eachsubstrate molecule to the protein is an essential prelude. In the simplest case, ifwe denote the enzyme by E, the substrate by S, and the product by P, the basicreaction path is E + S → ES → EP → E + P. There is a limit to the amount of substrate that a single enzyme molecule can process in a given time.
Although anincrease in the concentration of substrate increases the rate at which product isformed, this rate eventually reaches a maximum value (Figure 3–46). At that pointthe enzyme molecule is saturated with substrate, and the rate of reaction (Vmax)depends only on how rapidly the enzyme can process the substrate molecule. Thismaximum rate divided by the enzyme concentration is called the turnover number. Turnover numbers are often about 1000 substrate molecules processed persecond per enzyme molecule, although turnover numbers between 1 and 10,000are known.The other kinetic parameter frequently used to characterize an enzyme is itsKm, the concentration of substrate that allows the reaction to proceed at one-halfits maximum rate (0.5 Vmax) (see Figure 3–46).
A low Km value means that theenzyme reaches its maximum catalytic rate at a low concentration of substrate andgenerally indicates that the enzyme binds to its substrate very tightly, whereas ahigh Km value corresponds to weak binding. The methods used to characterizeenzymes in this way are explained in Panel 3–2 (pp. 142–143).Enzymes Speed Reactions by Selectively Stabilizing TransitionStatesEnzymes achieve extremely high rates of chemical reaction—rates that are farhigher than for any synthetic catalysts.
There are several reasons for this efficiency. First, when two molecules need to react, the enzyme greatly increases thelocal concentration of both of these substrate molecules at the catalytic site, holding them in the correct orientation for the reaction that is to follow. More importantly, however, some of the binding energy contributes directly to the catalysis.Substrate molecules must pass through a series of intermediate states of alteredgeometry and electron distribution before they form the ultimate products of thereaction.
The free energy required to attain the most unstable intermediate state,called the transition state, is known as the activation energy for the reaction, andit is the major determinant of the reaction rate. Enzymes have a much higheraffinity for the transition state of the substrate than they have for the stable form.Figure 3–46 Enzyme kinetics. The rateof an enzyme reaction (V) increases asthe substrate concentration increasesuntil a maximum value (Vmax) is reached.At this point all substrate-binding sites onthe enzyme molecules are fully occupied,and the rate of reaction is limited bythe rate of the catalytic process on theenzyme surface. For most enzymes,the concentration of substrate at whichthe reaction rate is half-maximal (Km) isa measure of how tightly the substrateis bound, with a large value of Kmcorresponding to weak binding.142PANEL 3–2: Some of the Methods Used to Study EnzymesWHY ANALYZE THE KINETICS OF ENZYMES?Enzymes are the most selective and powerful catalysts known.An understanding of their detailed mechanisms provides acritical tool for the discovery of new drugs, for the large-scaleindustrial synthesis of useful chemicals, and for appreciatingthe chemistry of cells and organisms.
A detailed study of therates of the chemical reactions that are catalyzed by a purifiedenzyme—more specifically how these rates change withchanges in conditions such as the concentrations of substrates,products, inhibitors, and regulatory ligands—allowsbiochemists to figure out exactly how each enzyme works.For example, this is the way that the ATP-producing reactionsof glycolysis, shown previously in Figure 2–48, weredeciphered—allowing us to appreciate the rationale for thiscritical enzymatic pathway.In this Panel, we introduce the important field of enzymekinetics, which has been indispensable for deriving much ofthe detailed knowledge that we now have about cellchemistry.STEADY-STATE ENZYME KINETICSMany enzymes have only one substrate, which they bind andthen process to produce products according to the schemeoutlined in Figure 3–50A.
In this case, the reaction is written asE+Sk1k –1ESkcatE+PHere we have assumed that the reverse reaction, in which E + Precombine to form EP and then ES, occurs so rarely that we canignore it. In this case, EP need not be represented, and we canexpress the rate of the reaction—known as its velocity, V, asAt this steady state, [ES] is nearly constant, so thatrate of ES breakdownk–1 [ES] + kcat [ES]or, since the concentration of the free enzyme, [E], is equalto [Eo] – [ES],[ES] =V = kcat [ES]where [ES] is the concentration of the enzyme–substrate complex,and kcat is the turnover number, a rate constant that has a valueequal to the number of substrate molecules processed perenzyme molecule each second.But how does the value of [ES] relate to the concentrations thatwe know directly, which are the total concentration of theenzyme, [Eo], and the concentration of the substrate, [S]? Whenenzyme and substrate are first mixed, the concentration [ES] willrise rapidly from zero to a so-called steady-state level, asillustrated below.rate of ES formationk1 [E][S]=k1k–1 + kcat[E][S]=k1k–1 + kcat[Eo] – [ES] [S]Rearranging, and defining the constant Km ask–1 + kcatk1we get[ES] =[Eo][S]Km + [S]or, remembering that V = kcat [ES], we obtain the famousMichaelis–Menten equationconcentrations[S][P]V =[Eo][ES][E]0timesteady state:ES almost constantKm + [S]As [S] is increased to higher and higher levels, essentially all ofthe enzyme will be bound to substrate at steady state; at thispoint, a maximum rate of reaction, Vmax , will be reached whereV = Vmax = kcat [Eo].
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